The overall goal of this project is to develop novel and improved cord blood expansion strategies and to test in a clinically relevant large animal model whether these strategies can overcome current limitations of cord blood transplantation. Despite some important advantages of cord blood, such as rapid accessibility and a low risk of graft-versus-host disease (GVHD) even in a human leukocyte antigen (HLA) mismatched setting, a major limitation of cord blood transplantation has been the limited number of cord blood cells available, particularly for adult patients. Thus, delayed engraftment and infectious complications have remained significant concerns with cord blood transplantation even with the recently described transplantation of two partially matched cord blood units. Cord blood expansion has been proposed to improve engraftment and hematopoietic reconstitution. However, expansion strategies using cytokines only have so far not resulted in significant improvements in cord blood engraftment in clinical studies. The use of stem cell self-renewal genes has been proposed, and studies in the mouse have shown that overexpression of the transcription factor HOXB4 can result in a dramatic expansion of cord blood and adult stem cells. We have studied the use of HOXB4 for stem cell self-renewal and expansion in a nonhuman primate transplantation model. Using a competitive repopulation assay we directly compared engraftment of marrow-derived CD34+ cells expanded for up to 12 days with and without retrovirally expressed HOXB4. We found that engraftment, in particular short-term engraftment, was significantly higher with HOXB4-transduced / expanded CD34+ cells compared to cells expanded without HOXB4 under otherwise identical conditions. HOXB4 overexpression also resulted in a marked expansion of nonhuman primate cord blood cells. Thus, we propose in the current application to study HOXB4-mediated cord blood expansion and evaluate in a nonhuman primate transplantation model whether this strategy can result in improved engraftment and hematopoietic reconstitution. We propose to 1) optimize HOXB4-mediated cord blood expansion in vitro using retrovirally expressed HOXB4, 2) evaluate improved expansion / transplantation strategies in a nonhuman primate cord blood transplantation model, and 3) determine whether HOXB4-mediated expansion can be achieved with a novel TAT-HOXB4 fusion protein. Studying these strategies in a clinically relevant nonhuman primate model will allow for an efficient evaluation of safety and efficacy and facilitate a rapid translation of our findings to clinical cord blood expansion / transplantation protocols.